Transgenic Plants

ABSTRACT

Disclosed herein are transgenic plants having recombinant DNA which expresses a G1073 transcription factor which provides enhanced resistance and/or tolerance to water deficit. More specifically the DNA constructs comprise a polynucleotide which encodes at least a functional part of a G1073 transcription factor or a homologous transcription factor.

REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/783,710, filed Feb. 21, 2004 and incorporated herein by reference inits entirety, which claims priority to U.S. Provisional Application60/449,054 filed Feb. 22, 2003, incorporated herein by reference in itsentirety.

JOINT RESEARCH STATEMENT

The claimed invention, in the field of functional genomics and thecharacterization of plant genes for the improvement of plants, was madeby or on behalf of Mendel Biotechnology, Inc. and Monsanto Company as aresult of activities undertaken within the scope of a joint researchagreement in effect on or before the date the claimed invention wasmade.

INCORPORATION OF SEQUENCE LISTING

The sequences in the enclosed Sequence Listing are identical to thesequences in the Sequence Listing and computer readable form of priorU.S. Provisional Application 60/449,054 filed Feb. 22, 2003, whichcontain the file named “G1073FINAL.ST25.txt” which is 21 kb and wascreated on 21 Feb. 2003 and which is incorporated herein by reference.

FIELD OF THE INVENTION

Disclosed herein are DNA useful for producing transgenic plants andseeds and methods of making and using such transgenic plants and seed.

BACKGROUND OF THE INVENTION

Water deficit can have adverse effects on plants such as yieldreductions, increased susceptibility to disease and pests, reduced plantgrowth and reproductive failure. An object of this invention is toprovide plants which can express genes to ameliorate the adverse effectsof water deficit. Useful genes for expression especially during waterdeficit are genes which promote aspects of plant growth or fertility,genes which impart disease resistance, genes which impart pestresistance, and the like.

Considering the complexity of water use in land plants, especiallyduring conditions that produce water deficit, relatively few genesspecifically associated with this aspect of physiology have beenidentified. It would be of benefit to the art to increase the number andvariety of genes involved in regulating water use in plants, moreparticularly, in corn plants, and even more particularly in corn plantsexperiencing water deficit. It would be especially advantageous toidentify transcription factors which can be used in directing theproduction of proteins which are beneficial to the plant when producedduring water deficit.

Transcription factors are investigated for improving plant propertiesand traits in transgenic plants. Reference is made to WO 02079403 ofMendel Biotechnology, Inc. which claims priority from U.S. applicationSer. No. 09/823,676 (incorporated herein by reference) for a disclosureof a variety of Arabidopsis thaliana transcription factors including oneidentified as G1073 which are alleged to be useful for modifying plantbiomass, for methods of building DNA constructs which express transcriptfactors, and for methods of producing transformed plants with DNAconstructs which express transcription factors.

One of the goals of plant genetic engineering is to produce plants withagronomically, horticulturally or economically important traitsincluding tolerance to any of a variety of environmental stresses suchas water deficit. Many transgenic crop plants have recombinant DNA thatconfers herbicide and/or pest resistance traits. Incorporation ofadditional recombinant DNA for conferring crop improvement traits incrop plants presents a challenge of using DNA constructs of increasedcomplexity.

SUMMARY OF THE INVENTION

We have discovered that transcription factors G1073 and homologs (G1073transcription factors) are useful for imparting enhanced resistanceand/or tolerance to water deficit in transgenic plants. The presentinvention is directed to DNA which encode at least a functional part ofa G1073 transcription factor which is useful in transgenic plants forenhancing yield when the plants are subjected to water deficit. Oneaspect of this invention provides methods for providing transgenicplants with an enhanced resistance and/or tolerance to water deficit.More particularly the method comprises transforming plants withrecombinant DNA construct comprising DNA which encodes at least afunctional part of a G1073 transcription factor, e.g. which impartsresistance to and/or tolerance to water deficit. Another aspect of theinvention provides transgenic seed for growing a plant which isresistant to water deficit as compared to wild type wherein the genomeof said seed comprises recombinant DNA which expresses at least afunctional part of such a G1073 transcription factor, e.g. having anamino acid sequence comprising at least 50 contiguous amino acids of aG1073 transcription factor. In another aspect of the invention suchtransgenic seed has in its genome recombinant DNA which expresses atranscription factor polypeptide having an amino acid sequences which isat least 50% identical (and preferably of higher identity) with asynthetic consensus amino acid sequence from a conserved region of aG1073 transcription factor. In one aspect of the invention therecombinant DNA is exogenous DNA. In another aspect of the invention theDNA expressing a G1073 transcription factor is DNA from the Arabidopsisthaliana transcription factor G1073. In yet another aspect of theinvention the DNA expressing a G1073 transcription factor is not derivedfrom the Arabidopsis thaliana transcription factor G1073, but rather isderived from DNA expressing a homologous G1073 transcription factor fromanother species.

This invention also provides plants grown from such transgenic seed withrecombinant DNA expressing a G1073 transcription factor. Transformedplants with tolerance and/or resistance to water deficit shouldinherently provide enhanced yield as compared to wild type plants whichare stunted by or succumb to water deficit. One aspect of the inventionprovides transgenic plants with stacked engineered traits, e.g. a cropimprovement trait provided by recombinant DNA expressing a G1073transcription factor in combination with herbicide and/or pestresistance traits.

Another aspect of the invention provides hybrid corn with stackedengineered traits. One embodiment of such hybrid corn is the progeny ofa transgenic ancestor corn plant having in its genome a recombinant DNAwhich expresses a G1073 transcription factor in combination with anherbicide and/or pest resistance trait. Embodiments of such hybrid cornhave a transgenic male ancestor corn plant which has in its genomerecombinant DNA which confers herbicide resistance and/or pestresistance.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an amino acid sequence alignment of:

(a) all of SEQ ID NO:7 representing conserved amino acid sequence in anArabidopsis transcription factor denoted “G1067” which is disclosed inapplication Ser. No. 09/934,455;(b) all of SEQ ID NO:8 representing conserved amino acid sequence in anArabidopsis transcription factor denoted “G1073” which has the fullamino acid sequence of SEQ ID NO:1;(c) residues 1-59 and 67-106 of SEQ ID NO:9 representing conserved aminoacid sequence in a cotton transcription factor which has the full aminoacid sequence of SEQ ID NO:3;(d) residues 1-59 and 69-108 of SEQ ID NO:10 representing conservedamino acid sequence in a rice transcription factor which has the fullamino acid sequence of SEQ ID NO:2; and(e) all of SEQ ID NO: 11 which is a consensus representation of thoseconserved amino acid sequences.

DETAILED DESCRIPTION OF THE INVENTION

As used herein a “G1073 transcription factor” means a protein which isexpressed by DNA of SEQ ID NO:4-6 and a protein having the amino acidsequence of SEQ ID NO:1-3 and a protein having the conserved amino acidsequence of SEQ ID NO:7-10 and a protein having the consensus amino acidsequence of SEQ ID NO:11 and a homologue protein from another speciesand parts of such proteins that function to provide thewater-deficit-tolerance trait exhibited in Arabidopsis thaliana, e.g. inthe assay illustrated in the example below.

As used herein “water deficit” is a plant condition characterized bywater potential in a plant tissue of less than −0.7 megapascals (MPa),e.g. −0.8 Mpa. Water potential in maize is conveniently measured byclamping a leaf segment in a pressurizable container so that a cut crosssection of leaf is open to atmospheric pressure. Gauge pressure (aboveatmospheric pressure) on the contained leaf section is increased untilwater begins to exude from the atmospheric-pressure-exposed crosssection; the gauge pressure at incipient water exudation is reported asnegative water potential in the plant tissue, e.g. 7 bars of gaugepressure is reported as −0.7 MPa water potential. Water deficit can beinduced by withholding water from plants for sufficient time that wildtype plants are deleteriously affected, e.g. as manifested by reducedyield, stunted growth, retarded development, death or the like. Theplants of this invention show a remarkable risibility after periods ofwater deficit that are adverse to wild type plants.

As used herein “yield” of a crop plant means the production of a crop,e.g. shelled corn kernels or soybean or cotton fiber, per unit ofproduction area, e.g. in bushels per acre or metric tons per hectare,often reported on a moisture adjusted basis, e.g. corn is typicallyreported at 15.5% moisture. Moreover a bushel of corn is defined by lawin the State of Iowa as 56 pounds by weight, a useful conversion factorfor corn yield is: 100 bushels per acre is equivalent to 6.272 metrictons per hectare. Other measurements for yield are in common practice.As used herein a “transgenic” organism, e.g. plant or seed, is one whosegenome has been altered by the incorporation of exogenous geneticmaterial or additional copies of native genetic material, e.g. bytransformation or recombination of the organism or an ancestor organism.Transgenic plants include progeny plants of an original plant derivedfrom a transformation process including progeny of breeding transgenicplants with wild type plants or other transgenic plants. Crop plants ofinterest in the present invention include, but are not limited to soy,cotton, canola, maize, wheat, sunflower, sorghum, alfalfa, barley,millet, rice, tobacco, fruit and vegetable crops, and turfgrass.

As used herein an “herbicide resistance” trait is a characteristic of atransgenic plant that is resistant to dosages of an herbicide that istypically lethal to a progenitor plant. Such herbicide resistance canarise from a natural mutation or more typically from incorporation ofrecombinant DNA that confers herbicide resistance. Herbicides for whichresistance is useful in a plant include glyphosate herbicides,phosphinothricin herbicides, oxynil herbicides, imidazolinoneherbicides, dinitroaniline herbicides, pyridine herbicides, sulfonylureaherbicides, bialaphos herbicides, sulfonamide herbicides andgluphosinate herbicides. To illustrate that the production of transgenicplants with herbicide resistance is a capability of those of ordinaryskill in the art reference is made to U.S. patent applicationpublications 2003/0106096A1 and 2002/0112260A1 and U.S. Pat. Nos.5,034,322; 6,107,549 and 6,376,754, all of which are incorporated hereinby reference.

As used herein an “pest resistance” trait is a characteristic of atransgenic plant is resistant to attack from a plant pest such as avirus, a nematode, a larval insect or an adult insect that typically iscapable of inflicting crop yield loss in a progenitor plant. Such pestresistance can arise from a natural mutation or more typically fromincorporation of recombinant DNA that confers pest resistance. Forinsect resistance, such recombinant DNA can, for example, encode aninsect lethal protein such as a delta endotoxin of Bacillusthuringiensis bacteria or be transcribed to a dsRNA targeted forsuppression of an essential gene in the insect. To illustrate that theproduction of transgenic plants with pest resistance is a capability ofthose of ordinary skill in the art reference is made to U.S. Pat. Nos.5,250,515 and 5,880,275 which disclose plants expressing an endotoxin ofBacillus thuringiensis bacteria, to U.S. Pat. No. 6,506,599 whichdiscloses control of invertebrates which feed on transgenic plants whichexpress dsRNA for suppressing a target gene in the invertebrate, to U.S.Pat. No. 5,986,175 which discloses the control of viral pests bytransgenic plants which express viral replicase, and to U.S. PatentApplication Publication 2003/0150017 A1 which discloses control of pestsby a transgenic plant which express a dsRNA targeted to suppressing agene in the pest, all of which are incorporated herein by reference.

SEQ ID NO: 1 provides the amino acid sequence of Arabidopsis thalianatranscription factor G1073, which are disclosed in U.S. application Ser.No. 09/823,676, filed Mar. 26, 2001, incorporated herein by reference.

SEQ ID NO:2 provides the amino acid sequence of part of the rice (Oryzasativa) polypeptide which is a homolog of the Arabidopsis thaliana G1073transcription factor.

SEQ ID NO:3 provides the amino acid sequence of part of the cotton(Gossypium hirsutum) polypeptide which is a homolog of the Arabidopsisthaliana G1073 transcription factor.

SEQ ID NO:4 provides DNA from the gene encoding an Arabidopsis thalianaG1073 transcription factor of SEQ ID NO:1.

SEQ ID NO:5 provides DNA from the gene encoding a rice transcriptionfactor of SEQ ID NO:2.

SEQ ID NO:6 provides DNA from the gene encoding a cotton transcriptionfactor of SEQ ID NO:3.

SEQ ID NO:7 provides a conserved region of the amino acid sequence ofthe Arabidopsis thaliana transcription factor G1067 which is disclosedin U.S. application Ser. No. 09/934,455, incorporated herein byreference.

SEQ ID NO: 8 provides a conserved region of the amino acid sequence ofthe Arabidopsis thaliana transcription factor G1073 (SEQ ID NO:1).

SEQ ID NO:9 provides a conserved region of the amino acid sequence ofthe cotton transcription factor (SEQ ID NO:3).

SEQ ID NO:10 provides a conserved region of the amino acid sequence ofthe rice transcription factor (SEQ ID NO:2).

SEQ ID NO:11 is an synthetic consensus amino acid sequence developedfrom alignment of the conserved region of SEQ ID NO: 7 through 10. Thealignment is illustrated in FIG. 1.

SEQ ID NO: 12 provides DNA from the gene encoding an Arabidopsisthaliana G1067 transcription factor, a conserved region of which is SEQID NO:7.

Polynucleotides of the present invention are DNA that is used to impartthe desired agronomic trait, e.g. such biological properties byproviding for enhanced protein activity in a transgenic plants byoverexpression of the polynucleotide, e.g. with a constitutive promoteror a promoter which is active during water deficit.

Protein and Polypeptide Molecules—Proteins of the present invention arewhole proteins or at least a sufficient portion of the protein to impartthe relevant biological activity of the protein, e.g. resistance and/ortolerance to water deficit in transgenic plants as compared to wildtype, as provided by constitutive expression of the Arabidopsis thalianaG1073 transcription factor or a functionally homologous transcriptionfactor. The term “protein” also includes molecules consisting of one ormore polypeptide chains. Thus, a polypeptide useful in the presentinvention may constitute an entire gene product or one or morefunctional portion of a natural protein which provides the agronomictrait of this invention, i.e. enhanced yield despite exposure to, waterdeficit.

Homologs of the polypeptides of the present invention may be identifiedby comparison of the amino acid sequence of the polypeptide to aminoacid sequences of polypeptides from the same or different plant sources,e.g. manually or by using known homology-based search algorithms such asthose commonly known and referred to as BLAST, FASTA, andSmith-Waterman.

A further aspect of the invention comprises functional homolog proteinswhich differ in one or more amino acids from those of a polypeptideprovided herein as the result of one or more of the well-knownconservative amino acid substitutions, e.g. valine is a conservativesubstitute for alanine and threonine is a conservative substitute forserine. Conservative substitutions for an amino acid within the nativepolypeptide sequence can be selected from other members of a class towhich the naturally occurring amino acid belongs. Representative aminoacids within these various classes include, but are not limited to: (1)acidic (negatively charged) amino acids such as aspartic acid andglutamic acid; (2) basic (positively charged) amino acids such asarginine, histidine, and lysine; (3) neutral polar amino acids such asglycine, serine, threonine, cysteine, tyrosine, asparagine, andglutamine; and (4) neutral nonpolar (hydrophobic) amino acids such asalanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, and methionine. Conserved substitutes for an amino acidwithin a native amino acid sequence can be selected from other membersof the group to which the naturally occurring amino acid belongs. Forexample, a group of amino acids having aliphatic side chains is glycine,alanine, valine, leucine, and isoleucine; a group of amino acids havingaliphatic-hydroxyl side chains is serine and threonine; a group of aminoacids having amide-containing side chains is asparagine and glutamine; agroup of amino acids having aromatic side chains is phenylalanine,tyrosine, and tryptophan; a group of amino acids having basic sidechains is lysine, arginine, and histidine; and a group of amino acidshaving sulfur-containing side chains is cysteine and methionine.Naturally conservative amino acids substitution groups are:valine-leucine, valine-isoleucine, phenylalanine-tyrosine,lysine-arginine, alanine-valine, aspartic acid-glutamic acid, andasparagine-glutamine. A further aspect of the invention comprisespolypeptides which differ in one or more amino acids from those of adescribed protein sequence as the result of deletion or insertion of oneor more amino acids in a native sequence.

Polypeptides of the present invention that are variants of thepolypeptides provided herein will generally demonstrate significantidentity with the polypeptides provided herein. Of particular interestare polypeptides having at least 50% sequence identity, more preferablyat least about 70% sequence identity or higher, e.g. at least about 80%sequence identity with (a) an synthetic consensus amino acid sequence ofSEQ ID NO:11 or (b) a conserved amino acid region of SEQ ID NO: 7through 10 or (c) an amino acid sequence of SEQ ID NO:1 through 3, or(d) other functional homologs of any polypeptide identified in (a)through (c). Of course useful polypeptides also include those withhigher identity to such a polypeptide sequence, e.g. 90%, to 100%identity. Other polypeptides of interest have at least 50 or more, e.g.at least 60 or 70 of the amino acids of a conserved segment of thetranscription factors proteins as defined by SEQ ID NO:7 through 10 andthe synthetic consensus amino acid sequence of SEQ ID NO:11. Of courseuseful polypeptides also include those with higher percentage of theamino acids in an protein segment of SEQ ID NO:7 thorough 11.

Recombinant DNA Constructs—The present invention contemplates the use ofpolynucleotides which encode a protein effective for impartingresistance and/or tolerance to water deficit in plants. Suchpolynucleotides are assembled in recombinant DNA constructs usingmethods known to those of ordinary skill in the art. A useful technologyfor building DNA constructs and vectors for transformation is theGATEWAY™ cloning technology (available from Invitrogen LifeTechnologies, Carlsbad, Calif.) uses the site specific recombinase LRcloning reaction of the Integrase/att system from bacterophage lambdavector construction, instead of restriction endonucleases and ligases.The LR cloning reaction is disclosed in U.S. Pat. Nos. 5,888,732 and6,277,608, U.S. Patent Application Publications 2001283529, 2001282319and 20020007051, all of which are incorporated herein by reference. TheGATEWAY™ Cloning Technology Instruction Manual which is also supplied byInvitrogen also provides concise directions for routine cloning of anydesired RNA into a vector comprising operable plant expression elements.

Transgenic DNA constructs used for transforming plant cells willcomprise the heterologous DNA which one desires to introduced into and apromoter to express the heterologous DNA in the host maize cells. As iswell known in the art such constructs typically also comprise a promoterand other regulatory elements, 3′ untranslated regions (such aspolyadenylation sites), transit or signal peptides and marker geneselements as desired. For instance, see U.S. Pat. Nos. 5,858,642 and5,322,938 which disclose versions of the constitutive promoter derivedfrom cauliflower mosaic virus (CaMV35S), U.S. Pat. No. 6,437,217 whichdiscloses a maize RS81 promoter, U.S. Pat. No. 5,641,876 which disclosesa rice actin promoter, U.S. Pat. No. 6,426,446 which discloses a maizeRS324 promoter, U.S. Pat. No. 6,429,362 which discloses a maize PR-1promoter, U.S. Pat. No. 6,232,526 which discloses a maize A3 promoter,U.S. Pat. No. 6,177,611 which discloses constitutive maize promoters,U.S. Pat. No. 6,433,252 which discloses a maize L3 oleosin promoter,U.S. Pat. No. 6,429,357 which discloses a rice actin 2 promoter andintron, U.S. Pat. No. 5,837,848 which discloses a root specificpromoter, U.S. Pat. No. 6,084,089 which discloses cold induciblepromoters, U.S. Pat. No. 6,294,714 which discloses light induciblepromoters, U.S. Pat. No. 6,140,078 which discloses salt induciblepromoters, U.S. Pat. No. 6,252,138 which discloses pathogen induciblepromoters, U.S. Pat. No. 6,175,060 which discloses phosphorus deficiencyinducible promoters, U.S. Patent Application Publication 2002/0192813A1which discloses 5′, 3′ and intron elements useful in the design ofeffective plant expression vectors, U.S. patent application Ser. No.09/078,972 which discloses a coixin promoter, U.S. patent applicationSer. No. 09/757,089 which discloses a maize chloroplast aldolasepromoter, all of which are incorporated herein by reference.

In many aspects of the invention it is preferred that the promoterelement in the DNA construct should be capable of causing sufficientexpression to result in the production of an effective amount of thetranscription factor in water deficit conditions. Such promoters can beidentified and isolated from the regulatory region of plant genes whichare over expressed in water deficit conditions. Specificwater-deficit-inducible promoters for use in this invention are derivedfrom the 5′ regulatory region of genes identified as a heat shockprotein 17.5 gene (HSP17.5), an HVA22 gene (HVA22), and a cinnamic acid4-hydroxylase (CA4H) gene (CA4H) of Zea maize. Suchwater-deficit-inducible promoters are disclosed in U.S. provisionalapplication Ser. No. 60/435,987, filed Dec. 20, 2002, incorporatedherein by reference.

In general it is preferred to introduce heterologous DNA randomly, i.e.at a non-specific location, in the plant genome. In special cases it maybe useful to target heterologous DNA insertion in order to achieve sitespecific integration, e.g. to replace an existing gene in the genome. Insome other cases it may be useful to target a heterologous DNAintegration into the genome at a predetermined site from which it isknown that gene expression occurs. Several site specific recombinationsystems exist which are known to function implants include cre-lox asdisclosed in U.S. Pat. No. 4,959,317 and FLP-FRT as disclosed in U.S.Pat. No. 5,527,695, both incorporated herein by reference.

Constructs and vectors may also include a transit peptide for targetingof a gene target to a plant organelle, particularly to a chloroplast,leucoplast or other plastid organelle. For a description of the use of achloroplast transit peptide see U.S. Pat. No. 5,188,642, incorporatedherein by reference.

In practice DNA is introduced into only a small percentage of targetcells in any one experiment. Marker genes are used to provide anefficient system for identification of those cells that are stablytransformed by receiving and integrating a transgenic DNA construct intotheir genomes. Preferred marker genes provide selective markers whichconfer resistance to a selective agent, such as an antibiotic orherbicide. Potentially transformed cells are exposed to the selectiveagent. In the population of surviving cells will be those cells where,generally, the resistance-conferring gene has been integrated andexpressed at sufficient levels to permit cell survival. Cells may betested further to confirm stable integration of the exogenous DNA.Useful selective marker genes include those conferring resistance toantibiotics such as kanamycin (nptII), hygromycin B (aph IV) andgentamycin (aac3 and aacC4) or resistance to herbicides such asglufosinate (bar or pat) and glyphosate (EPSPS). Examples of suchselectable are illustrated in U.S. Pat. Nos. 5,550,318; 5,633,435;5,780,708 and 6,118,047, all of which are incorporated herein byreference. Screenable markers which provide an ability to visuallyidentify transformants can also be employed, e.g., a gene expressing acolored or fluorescent protein such as a luciferase or green fluorescentprotein (GFP) or a gene expressing a beta-glucuronidase or uidA gene(GUS) for which various chromogenic substrates are known.

Transformation Methods and Transgenic Plants—Methods and compositionsfor transforming plants by introducing a transgenic DNA construct into aplant genome in the practice of this invention can include any of thewell-known and demonstrated methods. Preferred methods of planttransformation are microprojectile bombardment as illustrated in U.S.Pat. Nos. 5,015,580; 5,550,318; 5,538,880; 6,160,208; 6,399,861 and6,403,865 and Agrobacterium-mediated transformation as illustrated inU.S. Pat. Nos. 5,635,055; 5,824,877; 5,591,616; 5,981,840 and 6,384,301,all of which are incorporated herein by reference. See also U.S.application Ser. No. 09/823,676, incorporated herein by reference, for adescription of vectors, transformation methods, and production oftransformed Arabidopsis thaliana plants where transcription factors suchas G1073 are constitutively expressed by a CaMV35S promoter.

Transformation methods of this invention to provide plants with enhancedenvironmental stress tolerance are preferably practiced in tissueculture on media and in a controlled environment. “Media” refers to thenumerous nutrient mixtures that are used to grow cells in vitro, thatis, outside of the intact living organism. Recipient cell targetsinclude, but are not limited to, meristem cells, callus, immatureembryos and gametic cells such as microspores, pollen, sperm and eggcells. It is contemplated that any cell from which a fertile plant maybe regenerated is useful as a recipient cell. Callus may be initiatedfrom tissue sources including, but not limited to, immature embryos,seedling apical meristems, microspores and the like. Those cells whichare capable of proliferating as callus also are recipient cells forgenetic transformation. Practical transformation methods and materialsfor making transgenic plants of this invention, e.g. various media andrecipient target cells, transformation of immature embryos andsubsequent regeneration of fertile transgenic plants are disclosed inU.S. Pat. No. 6,194,636 and U.S. patent application Ser. No. 09/757,089,which are incorporated herein by reference.

The seeds of this invention can be harvested from fertile transgenicplants and be used to grow progeny generations of transformed plants ofthis invention including hybrid plants line comprising the DNA constructexpressing a transcription factor which provides the benefits ofresistance and/or tolerance to water deficit.

Breeding of Transgenic Plants

In addition to direct transformation of a plant with a recombinant DNAconstruct, transgenic plants can be prepared by crossing a first planthaving a recombinant DNA construct with a second plant lacking theconstruct. For example, recombinant DNA can be introduced into a plantline that is amenable to transformation to produce a transgenic plantwhich can be crossed with a second plant line to introgress therecombinant DNA into the second plant line.

In one aspect of the invention a transgenic plant with recombinant DNAconferring a crop improvement trait is crossed with a transgenic planthaving recombinant DNA conferring herbicide and/or pest resistance toproduce progeny plants having recombinant DNA that confers both the cropimprovement trait and the herbicide and/or pest resistance trait.Preferably, in such breeding for combining traits the transgenic plantdonating the crop improvement trait is a female line and the transgenicplant donating the herbicide and/or pest resistance trait is a maleline. The progeny of this cross will segregate such that some of theplant will carry the DNA for both parental traits and some will carryDNA for one parental trait; such plants can be identified by markersassociated with parental recombinant DNA Progeny plants carrying DNA forboth parental traits can be crossed back into the female parent linemultiple times, e.g. usually 6 to 8 generations, to produce a progenyplant with substantially the same genotype as one original transgenicparental line but for the recombinant DNA of the other transgenicparental line.

In yet another aspect of the invention hybrid transgenic seed, e.g. ahybrid transgenic corn seed, is produced by crossing a female transgeniccorn line containing recombinant DNA conferring a crop improvement traitwith a male transgenic corn line containing recombinant DNA conferringherbicide and/or pest resistance. In a preferred aspect of thisinvention hybrid transgenic corn seed is produced by crossing a femaletransgenic corn line with recombinant DNA conferring both a cropimprovement trait and herbicide resistance with a male transgenic cornline with recombinant DNA conferring both herbicide resistance and pestresistance.

Having now generally described the invention, the same will be morereadily understood through reference to the following example which isprovided by way of illustration, and is not intended to be limiting ofthe present invention, unless specified.

Examples

These examples illustrates the use of a polynucleotide encodingtranscription factor G1073 to provide a transgenic plant exhibitingenhanced tolerance for and/or resistance to growing conditions of waterdeficit.

Transgenic Arabidopsis thaliana was prepared with an exogenous DNAconstruct comprising a constitutive promoter of CaMV 35S operably linkedto a polynucleotide of SEQ ID NO: 12 encoding Arabidopsis thalianatranscription factor G1073 of SEQ ID NO:1. Transgenic and wild typeplants were potted in garden soil in a controlled environmental growthchamber in a 12 hour light/dark cycle. When the plants were at the earlyflowering stage, they were screened for water-deficit tolerance. Waterwas withheld until the wild type plants began wilting. Carbon dioxideassimilation rates were measured at growth and saturated conditions.Growth conditions were light at 200 μmol m⁻² s⁻¹ and 350 ppm CO₂.Saturating conditions were light at 1000 μmol m⁻² s⁻¹ and 1000 ppm CO₂.Wild type plants had smaller stomata conductance and lower CO₂assimilation rates than did the transgenic plants.

Transgenic soybean was prepared with an exogenous DNA constructcomprising a constitutive promoter CaMV35S operably linked to apolynucleotide of SEQ ID NO: 12 encoding Arabidopsis thalianatranscription factor G1073 of SEQ ID NO:1. When grown in water-deficitassay conditions the transgenic soybean showed enhanced resistanceand/or tolerance to water deficit as compared to wild type.

Transgenic corn was prepared with an exogenous DNA construct comprisinga constitutive promoter of the rice actin 1 gene operably linked to apolynucleotide of SEQ ID NO: 12 encoding Arabidopsis thalianatranscription factor G1073 of SEQ ID NO:1. Transgenic corn exhibitedvarious enhance traits, e.g. increased biomass, increased seed oil,increased yield and the ability to utilize high levels of nitrogen.

1. A transgenic seed for growing a transgenic plant having in its genomerecombinant DNA which expresses a transcription factor comprising anamino acid sequence with at least about 80% sequence identity to theamino acid sequence of SEQ ID NO: 2, wherein said transcription factorimparts tolerance to water deficit in a transgenic plant grown from saidseed.
 2. The transgenic seed of claim 1 wherein said transcriptionfactor comprises an amino acid sequence with at least about 90% sequenceidentity to the amino acid sequence of SEQ ID NO:2.
 3. The transgenicseed of claim 1 wherein said transcription factor comprises an aminoacid sequence with at least about 100% sequence identity to the aminoacid sequence of SEQ ID NO:2.
 4. The transgenic seed of claim 1, whereinsaid recombinant DNA comprises a sequence which is substantially thesame as SEQ ID NO:5.
 5. The transgenic seed of claim 1 wherein saidplant is a crop selected from the group consisting of a variety ofmaize, soybean, cotton, rice, wheat, canola and turfgrass.
 6. Atransgenic water-deficit tolerant transgenic plant grown from a seed ofclaim
 1. 7. A method for improving the yield of a crop plant variety ascompared to said crop variety lacking recombinant DNA expressing a G1073transcription factor when said crop varieties are grown in a waterdeficient environment, said method comprising inserting into the genomeof said variety recombinant DNA which expresses a transcription factorcomprising an amino acid sequence with at least about 80% sequenceidentity to the amino acid sequence of SEQ ID NO:
 2. 8. A method ofimproving a hybrid crop plant by crossing a first crop with a secondcrop wherein pollen from said first crop contains recombinant DNA whichexpresses a transcription factor comprising an amino acid sequence withat least about 80% sequence identity to the amino acid sequence of SEQID NO:
 2. 9. The method of claim 8 wherein one of said crops comprisesrecombinant DNA which expresses a protein that confers at least one ofan herbicide resistance trait or a pest resistance trait.
 10. A hybridcorn seed which is the progeny of (a) a transgenic female ancestor cornplant having in its genome a recombinant DNA which expresses atranscription factor comprising an amino acid sequence with at leastabout 80% sequence identity to the amino acid sequence of SEQ ID NO: 2;(b) a transgenic male ancestor corn plant having in its genome arecombinant DNA which confers at least one of an herbicide resistancetrait or a pest resistance trait.
 11. The hybrid corn seed of claim 10wherein said transgenic female ancestor corn plant further has in itsgenome recombinant DNA which confers herbicide resistance.
 12. Thehybrid corn seed of claim 11 wherein said transgenic male ancestor cornplant has in its genome recombinant DNA which confers both herbicideresistance and insect resistance.
 13. The hybrid corn seed of claim 11having resistance to at least one herbicide selected from the groupconsisting of a glyphosate herbicide, a phosphinothricin herbicide, anoxynil herbicide, an imidazolinone herbicide, a dinitroanilineherbicide, a pyridine herbicide, a sulfonylurea herbicide, a bialaphosherbicide, a sulfonamide herbicide and a gluphosinate herbicide.